The invention relates to test strips for the detection of equol in aqueous solutions by a color change, and methods and uses relating thereto.
Isoflavones, also referred to as isoflavonoids, are compounds of mostly yellow coloration, which are derivatives of isoflavones and thus flavonoids. Isoflavones are secondary compounds from plants, which, amongst others, play a role in the plants' defence from pathogens. The ground body of isoflavone is found in clover species. Some well-known isoflavones are daidzein, found as a glucoside of daidzin in soy flour, genistein from soy beans and red clover, prunetin from the bark of plum trees, biochanin A from chickpeas and clover, orobol, santal from sandle wood, red wood and other woods and pratensein from fresh red clover. The isoflavone daidzein [4′,7-dihydroxyisoflavone; 7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one] is found in soy and is part of many foods and dietary supplements.
The isoflavane equol [4′,7-dihydroxyisoflavane, 3-(4-hydroxyphenyl)-7-chromanol] is produced in the intestinal flora after consumption of daidzein. It is assumed that the conversion is carried out by streptococci, lactamid acid bacteria and bifido bacteria. Equol is not found in soy or any other plants. Equol is thus part of the group of secondary plant metabolites.
Equol has a mild estrogenic activity (0.1% of the activity of steroid-estrogens) and can bind to the estrogen receptors ERα and ERβ. Many beneficial effects are attributed to equol, for instance a cholesterol reducing effect, an anti-inflammatory effect, positive effects on breast cancer and on physiological changes after menopause. Equol also inhibits DHT production in males due to interactions with the 5α reductase. It is assumed that DHT is a cause in the forming of prostate cancer in males. Other benefits may be the treatment of male pattern baldness, acne and other problems related to DHT.
After the consumption of foods which are rich in daidzein, equol is detectable in blood and urine if a person is capable of producing equol. However, only about a third (Caucasian population) up to half (Japanese population) of humans can produce equol from daidzein. In humans who are capable of producing equol (“equol producers”), the positive effects of a soy-rich diet is more pronounced compared to humans, which are not capable of producing equol.
In order to predict whether an isoflavone-rich diet will be beneficial to a person, it is necessary to determine whether the person is an equol producer. A convenient way of determining this seems to be the detection of equol in urine. In the art, various methods for the detection of equol were described.
WO 2004/009035 A2 relates to various beneficial effects of equol. According to this document, equol is detected after purification by high pressure liquid chromatography (HPLC) by a subsequent analysis by mass spectroscopy. Thus, the method requires complicated and expensive instruments.
Another method for the detection of equol is disclosed in JP 2006-242602. The subject of this document is to provide a simplified method, which does not require HPLC or mass spectroscopy. In order to solve this problem, the inventors provide two methods. A first method is based on the separation of a sample with thin layer chromatography. The method requires a pre-treatment of the sample with enzymes for removing saccharides. After the chromatographic separation of the isoflavones, the plate is developed with a solvent. Subsequently, steps for visualizing the isoflavones are carried out, such as subjecting the plate to a steam of iodine. Although avoiding complicated instruments such as HPLC columns and mass spectrometers, the method is relatively complicated and requires chemicals which cannot be handled easily. A second method requires the cultivation of microorganisms and is also complicated.
The inventors of JP 2006-242602 subsequently published an article with the title “Rapid and Convenient Detection of Urinary Equol by Thin-layer Chromatography”; (J Nutr. Sci. Vitaminol. (Tokyo), 2007, 53(1); 43-7). The method requires the hydrolyzation of urine samples, purification by a reverse phase silica gel column and thin layer chromatography. The method is applied for discriminating equol producers.
Early studies relating to the detection of equol were published in the 1940ies and 1950ies. A method for the color detection of equol in the urine of mares was disclosed by Dirscherl (1940, Physiol. Chem., 264, 57-63). The formation of a red ring at the junction of nitric acid and an aqueous sample solution is indicative of the formation of equol. Other methods are disclosed, in which an equol precipitate is produced and dissolved or in which a solid is produced on a microscopic slide and observed under the microscope. The methods disclosed in this document require the equipment of a chemical laboratory or instruments such as microscopes.
The problems known in the art for the detection of equol thus are relatively complicated. They either require sophisticated and expensive instruments or at least the equipment of a chemical laboratory. Further, potentially dangerous chemicals, such as nitric acid or iodine, are used during the test.